Immunologic Difference between Hypersensitivity to Mosquito Bite and Hemophagocytic Lymphohistiocytosis Associated with Epstein-Barr Virus Infection Wen-I Lee1,2*., Jainn-Jim Lin3,6., Meng-Ying Hsieh3,4, Syh-Jae Lin2, Tang-Her Jaing1,5, Shih-Hsiang Chen5, Iou-Jih Hung5, Chao-Ping Yang5, Chin-Jung Chen7, Yhu-Chering Huang7, Shin-Pai Li8, Jing-Long Huang1,2* 1 Primary Immunodeficiency Care And Research (PICAR) Institute, Chang Gung Children’s Hospital, Taoyuan, Taiwan, 2 Department of Pediatrics, Division of Allergy Asthma and Rheumatology, Chang Gung Children’s Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan, 3 Graduate Institute of Medical Clinics, Chang Gung University College of Medicine, Taoyuan, Taiwan, 4 Department of Pediatrics, Division of Neurology, Chang Gung Children’s Hospital, Taoyuan, Taiwan, 5 Department Pediatrics, Division of Hematology/Oncology, Chang Gung Children’s Hospital, Taoyuan, Taiwan, 6 Department of Pediatrics, Division of Critical Care and Emergency Medicine, Chang Gung Children’s Hospital, Taoyuan, Taiwan, 7 Department of Pediatrics, Division of Infection, Chang Gung Children’s Hospital, Taoyuan, Taiwan, 8 Department of Microbiology and Immunology, Chang Gung University College of Medicine, Taoyuan, Taiwan
Abstract Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, virus-triggered immune disease. Hypersensitivity to mosquito bite (HMB), a presentation of Chronic Active Epstein-Barr Virus infection (CAEBV), may progress to HLH. This study aimed to investigate the immunologic difference between the HMB episodes and the HLH episodes associated with EBV infection. Immunologic changes of immunoglobulins, lymphocyte subsets, cytotoxicity, intracellular perforin and granzyme expressions, EBV virus load and known candidate genes for hereditary HLH were evaluated and compared. In 12 HLH episodes (12 patients) and 14 HMB episodes (4 patients), there were both decreased percentages of CD4+ and CD8+ and increased memory CD4+ and activated (CD2+HLADR+) lymphocytes. In contrast to HMB episodes that had higher IgE levels and EBV virus load predominantly in NK cells, those HLH episodes with virus load predominantly in CD3+ lymphocyte had decreased perforin expression and cytotoxicity that were recovered in the convalescence period. However, there was neither significant difference of total virus load in these episodes nor candidate genetic mutations responsible for hereditary HLH. In conclusion, decreased perforin expression in the HLH episodes with predominant-CD3+ EBV virus load is distinct from those HMB episodes with predominant-NK EBV virus load. Whether the presence of non-elevated memory CD4+ cells or activated lymphocytes (CD2+HLADR+) increases the mortality rate in the HLH episodes remains to be further warranted through larger-scale studies. Citation: Lee W-I, Lin J-J, Hsieh M-Y, Lin S-J, Jaing T-H, et al. (2013) Immunologic Difference between Hypersensitivity to Mosquito Bite and Hemophagocytic Lymphohistiocytosis Associated with Epstein-Barr Virus Infection. PLoS ONE 8(10): e76711. doi:10.1371/journal.pone.0076711 Editor: Micah Luftig, Duke University Medical Center, United States of America Received June 8, 2013; Accepted August 27, 2013; Published October 18, 2013 Copyright: ß 2013 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Chang-Gung Medical Research Progress Grant (CMRPG 450022, 490012, and 4B0051) and a National Science Council Grant (NSC99-2314-B-182-003-MY3 and 102-2314-B-182A-039-MY3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail:
[email protected] (W-IL);
[email protected] (J-LH) . These authors contributed equally to this work.
candidate mutations of SH2D1A/SAP, PRF1, UNC13D, STX11, STXBP2, XIAP, and ITK can inhibit the exocytotic process of polarization, docking, priming, and fusion in cytotoxic T/natural killer (NK) cells, subsequently lead to defective cytotoxicity and overwhelming HLH in some rare hereditary and sporadic cases [16–20]. Hypersensitivity to mosquito bite (HMB) is a unique feature characterized by bulla formation with intense erythema on mosquito-bitten sites, escar healing and systemic manifestations like fever, lymphadenopathy, and splenomegaly [21,22]. Around 70% of CAEBV patients present as the HMB episode (HMBCAEBV) and have the potential of developing fulminant HLH [23]. To understand the possible mechanisms of HMB transformation into fulminant HLH, we evaluated and compared immunologic
Introduction The Epstein-Barr virus (EBV) infects B cells through surface CD21 in healthy individuals who are often asymptomatic or may present as infectious mononucleosis (IM) [1]. The outgrowth of EBV-infected B cells is controlled by T help cells secreting interferon (IFN)-c and NK-mediated cytoxicity, and later destroyed by EBV-specific cytotoxic T lymphocytes [2,3]. Patients with chronic active EBV (CAEBV) infection may have IM-like chronic symptoms such as fever and lymphadenopathy, and serologic evidence of persistent EBV infection [4–9]. Moreover, CAEBV can be exacerbated into fulminant (catastrophic) hemophagocytic lymphohistiocytosis (HLH) [10–14] and present with cytopenia, coagulopathy, central nervous system symptoms, and lipid changes, aside from IM-like features [15]. Known
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1
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Onset Age (y)
Onset year (AD)
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3Y6M
10Y7M
1Y
2Y10M
6Y6M
ES2/F
ES3/M
ES4/M
ES5/F
ES6/F
2006
2006
2005
2001
1998
1995
2002
2005
1Y10M
1Y7M
5Y2M
11Y
EM3/M
EM4/F
EM5/F
EM6/M
2
+ (Hb = 7.8; PL = 35K) + (Hb = 8.2; PL = 20K) + (Hb = 8.3; Neu = 990; PL = 90K)
+ + +
+ (Hb = 7.0; PL = 26 K) + (Hb = 5.5; Neu = 384; PL = 93K) + (Hb = 7.9; Neu = 540; PL = 14K) + (Hb = 5.1; Neu = 36; PL = 70K) + (Hb = 8.4; PL = 49K) + (Hb = 8.3; Neu = 60; PL = 67K)
+ + + + + +
+ (Hb = 8.5; PL = 56K)
+ (Hb = 8.4; PL = 24K)
+
+ (Hb = 8.7; PL = 12K)
+
Cytopenia
+
Fever
4M
12Y
18 Y
H3/M
H4/M
12Y3M
H2/M
H1/M
2 2 2 2 2 2 2 2 2
+ + + + + + + +
2007
2012
2005
2006
2011
2006
2010
2011
+
2
+
2008
2005
2
+
2
+
2005
2006
2
+
2003
Hypersensitivity to Mosquito bite (HMB)-CAEBV
2005
2001
1993
6Y6M
EM2/M
1992
3Y2M
EM1/M
EBV-associated HLH (mortality)
6Y2M
ES1/M
EBV-associated HLH (survival)
Patient/ Sex
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
+
+
+
+
+
+
+
+
+
+
2/2
2/ND
2/2
ND/ND
ND/2
2/2
2/2
2/2
2/2
ND/ND
ND/ND
ND/ND
2/ND
2/+ (Fibri = 89)
+ (TG = 506)/ND
+ (TG = 473)/ + (Fibri = 47)
+ (TG = 295)/ND
2/+ (Fibri = 80)
ND/ND
2/2
2/ND
+ (TG = 293)/ 2
ND/ND
ND/ND
+ (TG = 332)/2
Hypertriglycemia Hemophago and/or cytosis hypofibrinogenemia
2/2
2/2
+/+
+/+
2/2
+/+
+/+
2/2
+/+
+/+
+/+
+/+
2/2
+/2
+/+
+/+
+/2
+/+
+/+
+/2
+/2
+/2
+/+
+/2
+/+
Splenomegaly/ Lymphadenopathy
2
2
2
2
2
2
2
+ (3479)
2
2
2
2
+ (2785)
2 (345)
2 (334)
+ (1543)
+ (14523)
+ (2134)
+ (7821)
+ (2404)
+ (751)
+ (13906)
+ (11788)
+ (3298)
+ (1479)
Ferritin*
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
+
+
ND
ND
+
+
+
ND
ND
+
Decreased NK activity
112/57
114/62
45/78
75/58
89/55
123/72
64/43
122/87
54/67
104/86
73/65
149/82
237/174
66/45
63/23
2109/485
358/253
2898/1285
471/204
342/214
2127/1665
603/358
16/10
762/170
326/118
AST/ ALT*
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
ST/NSAID
IVIG/CsA
IVIG/ST
IVIG
IVIG/ST/G-CSF
IVIG
IVIG/ST
IVIG/ST/CsA/VP16
IVIG/ST/CsA
IVIG/ST/CsA/VP16
ST
IVIG/ST
IVIG/ST/VP16
Treatment
15 days
58 days
13 days
29 days
8 days
14 days
Deceased after treatment
Table 1. Laboratory hematology, treatment, and prognosis of patients with hemophagocytic lymphohistiocytosis (HLH)* and hypersensitivity to mosquito bite (HMB) episodes related to EBV infection.
Distinction in EBV-Associated HBM and HLH Episodes
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Abbreviations: M, male; F, female; Hb, hemoglobin; PL, platelet; Neu, neutrophil; TG, triglyceride; Fribi; fibrinogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; IVIG, intravenous immunoglobulin; ST, steroid prednisolone or dexamethasone; CsA, cyclosporine A; VP-16, etoposide; ND, not done; NSAID, Non-steroidal anti-inflammatory drug; CAEBV, Chronic active EBV infection. *The diagnosis criteria included fever, splenomegaly, cytopenia (affecting 2 of 3 lineages, Hb ,9 mg/dL; PL ,100 K; and Neu ,1000), hyper-triglycerides (265 mg/dL) or hypo-fibrinogenemia (1.5 g/L), hemophagocytosis, lower or abscent NK-cell activity, ferritin .500 ul/L and soluble CD25.2400 U/ml. Twelve patients with HLH reached at least 5 criteria without detectable soluble CD25. a The normal range of fibrinogen, TG, ferritin, AST, and ALT was 190–380 mg/dL, ,150 mg/ml, 10–322 ng/ml, 13–40 U/L, and ,36 U/L, respectively. doi:10.1371/journal.pone.0076711.t001
ST/NSAID 137/69 2 2 +/+ 2/2 2 2 + 2012
Cytopenia Onset Age (y) Patient/ Sex
Table 1. Cont.
Onset year (AD)
Fever
Hypertriglycemia Hemophago and/or cytosis hypofibrinogenemia
Splenomegaly/ Lymphadenopathy
Ferritin*
Decreased NK activity
AST/ ALT*
Treatment
Deceased after treatment
Distinction in EBV-Associated HBM and HLH Episodes
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changes of immunoglobulins, lymphocyte subsets, cytotoxicity, intracellular perforin and granzyme expressions, EBV virus load and known candidate genes in patients with the episodes of HMBCAEBV and EBV-HLH.
Results Patients’ Characteristics During the 20-year period of 1993–2012, fourteen HMB episodes in 4 CAEBV patients (one female) and twelve HLH episodes in 12 patients (five females) associated with EBV infection (EBV-HLH) were studied in Table 1. The HMB episode could be a characteristic feature of CAEBV along with fever, lymphadenopathy or/and hepatosplenomagaly. At mosquito-bitten sites (Fig. 1), clear or/and hemorrhagic bulla with intense erythematous swelling typically occurred. They progressed into necrosis or ulcers, and healed with residual scarring as escar. In contrast to HMB-CAEBV episodes (the range of onset-age, 4 months-21 years; median, 12 years 3 months), acute EBV-HLH episodes (range, 1–11 years; median, 3 years 4 months) had cytopenia (Hb ,9.0 mg/dl and thrombocytopenia ,100,000/ mm3 in all; neutropenia ,1,000/mm3 in 5 patients), coagulopathy (abnormal PT, aPTT, D-dimmer, or fibrogen in 7 patinets), and atypical lymphocytes (over 10% in 3 patients). Both groups often had splenomegaly, lymphadenopathy, and varying degrees of elevated aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) levels. The main treatment regimens in patients with HLH episodes based on the HLH 2004 guidelines [15] included IVIG, steroids (prednisolone or dexamethasone), etoposide, and cyclosporine A. Six (50%) of 12 acute EBV-HLH patients who did not receive etoposide (VP16) and cyclosporine A treatment were mortalities. In four HMB-CAEBV patients who did not develop HLH episode to date, NSAID or steroids were given for febrile episodes. Serology studies for EBV in 26 episodes from 16 patients showed that 11 HLH patients (ES1-ES6 except ES4 and EM1EM6) had primary EBV infection with positive anti-VCA-IgM or/ and positive anti-EBEA ($160) (Table 2). One episode of HLH (patient ES4) and 14 HMB episodes (4 patients H1–H4) had mainly positive anti-VCA IgG and/or negative VCA IgM, suggestive of EBV reactivation. The EBV viral load detected by copy numbers in all episodes was $102.5 copies/ug, compatible with EBV activation [24]. In contrast to the HMB episodes with EBV copy number predominantly in NK cells, the HLH episodes had EBV virus load predominantly in lymphocytes (CD3+). Nonetheless, there was no significant difference in virus load of total lymphocytes among the HLH (survivors and fatal victims) and HMB episodes.
Immunoglobulin and Lymphocyte Sub-population In eight HLH episodes (8 patients) and 14 HMB episodes (4 patients), the basic immune function of immunoglobulins, lymphocyte sub-populations, memory cells, and activated lymphocytes revealed immune heterogeneity in each group. Based on the normal reference [25], relatively higher IgG, and/or IgA were present in two fatal HLH cases (patients EM3 and EM6), but not in HMB cases. The Trend of decreased percentages of total CD4+ but increased memory CD4+ and activated lymphocyte (CD2+HLADR+) was noted among survivors of the HLH and the HMB groups (Table 3).
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Distinction in EBV-Associated HBM and HLH Episodes
Figure 1. There were clear and hemorrhagic bulla with intense erythematous swelling at mosquito-bitten sites on the (A) right leg dorsum and (B) palm. Necrosis and ulcers clustered on the base of the toes and turned into escar formation after recovery. (C) Previous escar scar remitted and centrally dipped like a volcano on the left. doi:10.1371/journal.pone.0076711.g001
Discussion
Cytotoxicity to K562 Cell Lines and the Expression of Perforin and Granzyme
Without reaching the HLH diagnostic criteria, patients with the HMB episodes could have better prognosis than those with HLH episodes. Based on an updated international meeting for EBVassociated lympho-proliferative diseases (LPD) and the recent Kimura et al. study of 108 cases [24,26], EBV-associated LPD are defined to be overlapping umbrella syndromes and encompass five subgroups including CAEBV of T/NK-cell type (or systemic EBV plus T-cell LPD of childhood), HLH, severe mosquito bite allergy (or hypersensitivity to mosquito bite, HMB), hydroa vacciniforme (HV), and HV-like lymphoma [26]. Using these concepts to the patients here, four HMB patients initially presented as ‘‘severe mosquito bite allergy’’ without splenomegaly nor lymphadenopathy in the enrolled time, but gradually developed as CAEBV NK cell type with splenomegaly or/and lymphadenopathy after several HMB episodes. As noted in previous reports [26–30], patients with HMB have the potential to progress to HLH, and even lymphoma. To recognize the herald feature at an earlier stage by comparing HMB and HLH episodes, the HLH episodes with CD3-predominant virus load had lower perforin expression and thus related to impaired cytotoxicity that partially contributed to the development of HLH. In contrast, these HMB episodes with NK-predominant EBV virus load and higher IgE level had normal perforin expressions and did not reach a threshold to impair cytotoxicity. In HMB-CAEBV patients, higher IL-13 level, a Th2type cytokine, were detected and could induce the differentiation
Lymphocyte/NK cell cytotoxicity to K562 cells was evaluated in 8 HLH episodes (8 patients) and 14 HMB episodes (4 patients) during the febrile stage (Table 4). In contrast to those with HMB episodes, the eight HLH patients, of course, met more than five out of eight diagnostic criteria including decreased cytotoxicity, which returned to a normal range during the convalescent stage among survivors. Intracellular perforin rather than granzyme expression significantly diminished below the normal ranges in NK cells (TCRabCD56+ or CD16+56+) in the HLH episodes, but not in the HMB episodes (Table 4). The NKT (TCRab+CD56+) cells had similar results (data not shown). In the convalescent period, there were relatively lower perforin expressions in the four survivors of HLH despite being within the normal range. The HMB group had no such differences in cytotoxicity and in the expressions of perforin and granzyme.
Analysis of Candidate Genes All patients received genetic analysis for the PFR1, Mun13-4, STX11, STXBP2 and ITK genes. Eleven male patients had further SH2D1A/SAP and XIAP sequencing but no mutation was identified.
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Distinction in EBV-Associated HBM and HLH Episodes
Table 2. EBV serology and evidence in EBV-HLH and HMB-CAEBV episodes.
Patient (Year)
Serum antibodies of EBV profile
EBV Virus load log copies/ug genomic DNA
EBEA
EBNA
VCA IgG
VCA IgM
CD3+
CD16+CD56+
320
+
320
2
5.6
NA
EBV-HLH-Survival ES1 (1995) ES2 (1998)
NA
NA
320
+
3.9
2.8
ES3 (2001)
NA
NA
320
+
4.7
3.2
ES4 (2005)
80
+
640
2
4.2
2.3
ES5 (2006)
80
+
320
+
3.7
2.7
ES6 (2006)
80
2
320
+
4.9
2.4
EM1 (1992)
320
+
1280
+
NA
NA
EM2 (1993)
160
+
160
2
4.6
NA
EBV-HLH-Mortality
EM3 (2001)
NA
+
160
+
5.1
NA
EM4 (2002)
20
NA
NA
+
3.7
3.0
EM5 (2005)
20
2
320
+
4.5
3.2
EM6 (2005)
160
+
80
2
3.4
2.1
HMB-CAEBV H1 (2003)
80
20
640
2
3.1
5.2
(2005)
80
+
640
2
4.1
5.7
(2006)
160
+
320
2
NA
NA
(2008)
20
+
640
2
2.7
4.9
(2011)
20
2
1280
2
3.0
4.7
H2 (2005)
NA
+
2
+
2.4
5.1
(2007)
20
+
320
2
NA
NA
(2012)
20
+
640
2
3.9
4.7
H3 (2005)
160
+
2
+
2.9
3.7
(2006)
80
+
640
2
NA
NA
(2011)
80
+
1280
2
2.7
5.4
H4 (2006)
20
+
640
2
2.9
3.5
(2010)
20
+
160
2
NA
NA
(2012)
20
+
640
2
3.6
4.8
Abbreviations: EBV, Epstein-Barr virus; ENEA, Epstein-Barr virus early antigen; EBNA, Epstein-Barr virus nuclear antigen; VCA, viral capsid antigen; IgG, immunoglobulin G; IgM, immunoglobulin M; NA, not available. doi:10.1371/journal.pone.0076711.t002
Genetic defects of PFR1, Mun13-4, STX11, STXBP2, ITK (autosomal recessive), SH2D1A/SAP, and XIAP (X-linked) are responsible for hereditary HLH, with increased susceptibility to recurrent, and/or fatal EBV infection [15–20]. However, all are wild type in the study patients because of the conservative culture that discourage consanguineous marriage in our regions. Such findings are consistent with a recent genetic study from a cohort of 67 children of Chinese descent who had HLH and wild type candidate genes [34]. However, parallel to restored cytotoxicity after effective chemotherapy, perforin expression recovered but was maintained at the relatively lower border of the normal range. Single nucleotide polymorphism (SNP) of A91V and N252S in the PFR1 gene was found to decrease perforin expression and function that cause atypical HLH [35,36], but not identified of such SNP in our patients. This reflects that patients with borderline perforin expression may have decreased the perforin-granzyme B pathway to some extent, leading to insufficient apoptosis and subsequently developing HLH. EBV latent membrane protein 1 (LMP1) has been demonstrated to diminish SH2D1A expression and stronger
of B cells and enhance a class switch to IgE [23]. Whether the majority of EBV virus shifted from NK cells in the HBM status to T-lymphocytes after several HBM episodes and cytokine alternation, reached the threshold to weaken cytotoxicity and therefore cause HLH episode remains to be determined by additional study. The possibility of ‘‘shift’’ hypothesis was observed in a 35-yearold female patient who experienced at least eight HBM episodes since her first attack at the age of 21. Unfortunately, she succumbed to HLH acceleration in 1990 (beyond the study period of 1992–2012) [31]. Her EBV virus load evaluated by copy numbers from frozen PBMC revealed almost an equal amount in NK cells (103.4) and in T lymphocytes (103.1) during the HBM episodes, but eventually became more in T lymphocytes (104.2) than NK cells (103.6) in the HLH status after several HMB episodes [21,32]. Thus in Japan, CAEBV patients with the HBM episodes are encouraged to receive hematopoietic stem cell transplantation as early as possible if suitable donors are available [24,33], to prevent the process of HLH development and the oncogenic transformation of lymphoma [26–30].
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Distinction in EBV-Associated HBM and HLH Episodes
Table 3. Serum immunoglobulin values and lymphocyte subsets in HLH-EBV and HMB-CAEBV episodes related to EBV infection.
Absolute lymphocyte count
a
Immunoglobulin level (mg/dl)
Patient
IgM
IgA
IgG
Lymphocyte subsets percentages (%)b
IgE
CD4
CD8
CD19
Activated lymphocyte
CD16/56 Memory cell* B2
CD4+T2 EBV-associated survival ES1
1995
164
173
1190
79
ES4
2005
38
17
674
107
ES5
2006
42
59
867
42
ES6
2006
143
69
1495
147
32
1975 q 97
q
q
1432
34.2
18.4
13.2
8.9
31.6
7.8
2135
9.5
Q 14.8
71.5
q 3.8
51.6
q 1.4
4290
19.0
Q 11.2
3192
38.8
34.1
11.2
8
45.7
1547
32.4
18.5
11.2
10.8
9.4
Q 9.1
56.9
q 42.6
43.1 Q
q
23.7
q 3.6
65.1
q 18.9
34.1
q
EBV-associated dead EM3
2001
42
EM4
2002
32
46
568
56
446
EM5
2005
53
57
756
86
877
EM6
2007
45
576
q 2640 q 92
340
Q
Q
5.4
18.9
41.5
32.7
8.7
3.4
13.0
6.7
24.4
40.8
20.4
10.4
4.2
8.3
4.3
20.5
39.7
29.4
9.6
1.8
Q 10.2
2.3
Q
14.9
Hypersensitivity to Mosquito bite (HMB) H1
H2
H3
H4
2003
110
236
1650 q 1804
q
1874
14.9
Q 10.9
Q 11.5
59.0
q 48.2
q 9.1
48.1
q
2005
89
215
1756 q 1124
q
1945
19.4
Q 11.4
Q 14.8
47.2
q 54.2
q 10.2
47.5
q
2006
142
198
1324
2468
q
2147
22.4
Q 8.8
Q 21.0
39.6
q 44.5
q 16.5
61.4
q
2008
127
246
1942 q 2497
q
1258
27.3
Q 11.2
Q 19.7
38.7
q 39.7
q 11.8
50.9
q
2011
169
231
1237
1785
q
2013
33.7
12.1
Q 17.5
45.2
q 47.1
q 14.9
49.1
q
q
4984
2005
78
55
573
129
2007
NA
NA
NA
NA
2012
102
119
1745 q 952
2005
271
228
1420 q 1420
2006
NA
NA
NA
q
40.9
19.5
18.0
11.4
27.5
3278
24.2
Q 10.5
8.6
21.2
q 34.2
14.2
32.5
q
2846
32.5
11.4
9.7
24.8
q 39.5
q 19.4
24.1
q
3945
21.7
Q 13.7
5.4
Q 59.4
q 46.8
q 20.3
3125
24.5
Q 17.9
10.2
48.7
q 39.7
q 7.5
NA
7.2
14.3
q
66.1
2011
198
159
1328
897
q
2415
32.5
20.1
14.2
35.4
q 40.2
q 11.4
18.7
2006
115
242
1360
1260
q
3160
39.6
23.5
12.1
24.2
q 33.3
13.3
28.1
2010
129
214
1174
3145
q
2984
41.2
19.7
11.5
19.8
q 37.8
12.9
34.5
2012
147
119
1069
1694
q
2531
34.5
22.4
14.6
25.4
q 41.7
q 10.8
37.1
28–56
12–35
6–41
3–18
2–38
3–20
3–39
Normal range
q
14.3
Abbreviations: NA, not available; Q or q, below or above the normal range, respectively. a Normal ranges were from Stiehm RE. Immunologic Disorders in Infants and Children. 6th ed. Philadelphia, PA: Philadelphia Press, 2003. b Normal percentages were from Ref. 25. *Memory CD4+ T cell lymphocyte percentage = CD4+CD45RO+/CD4+CD45RO+ and CD4+CD45RO2; Memory CD19+ B cell lymphocyte percentage = CD19+CD27+/ CD19+CD27+ and CD19+CD272; Activated lymphocyte percentage = CD2+HLADR+/all lymphocytes. doi:10.1371/journal.pone.0076711.t003
augmentation to fight EBV infection and overcome cytokinesrelated catastrophic response in persistent and un-eradicated pathogen reactivation [38,40]. Thus, elevated activated lymphocytes and memory CD4+ cells recruit more effective response to suppress EBV activation and break down the process of overwhelming HLH. In dynamics of the whole CD4+ cell pool, the amount of CD4+ effector cells going apoptosis after activation was more than those turning to memory CD4+ cells and supplemental naı¨ve CD4+ cells. Consequently, the percentage of overall CD4+ cells trended to decrease but memory CD4+ to increase in survivors (in Table 3). However, lack of increased percentages of activated lymphocytes and memory CD4+ cells during the episode status in those fatal HLH patients implied that impending exhaustion of the whole T cell pool, challenged by EBV repeated activation, could be a warning sign of hematopoietic bone marrow failure, contributing to worse prognosis.
inflammation [37]. Whether the similar inhibitory effect of EBV infection-associated factors (LMP1 or other antigens derived from EBV) on the perforin expression is worth being investigated further. Notably in our patients, fatal HLH patients did not have elevated memory CD4+ cells and activated CD2+HLADR+ lymphocytes. Correlative to the differentiation and development of memory CD4+ cells and activated lymphocytes, T-cell receptor (TCR) in naı¨ve T cells recognized MHC class II (HLA-DR) on EBV-antigen-presenting cells (APC) and triggered signaling one pathway and subsequently programmed as T effector cells after additional stimulation from signal two or more accessory pathways [38,39]. Some subgroup of T effector cells that expressed activation molecules such as HLADR+, CD40L, ICOS, or CD69 and belonged to one pattern of the activated lymphocytes were able to continuously mature into memory T cells for robust
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Table 4. Cytotoxicity to K 562 cells and perforin and granzyme expressions in NK cells in the episodes and recovery status of EBVHLH and HBM-CAEBV.
Patient
Cytotoxicity to K562 cell lines
Perforin expression in NK cells
Effector to target cell ratio
(gated by CD56+TCRab2 or CD16+CD56+) (gated by CD56+TCRab2 or CD16+CD56+)
25/1
Percentage (mean fluorescent intensity)
Percentage (mean fluorescent intensity)
Recovery
Acute
Recovery
Acute
12.5/1
Acute
25/1
12.5/1
Granzyme expression in NK cells
Recovery
EBV-associated survival ES1
1995
22.4%
20.8%
42.4% NA
NA
57.8% (48.7619.6)
47.4% (54.4612.4)
56.7% (61.5618.7)
ES4
2005
18.6%
17.5%
39.7% 36.9%
39.1% (12.6620.0)
NA
49.7% (58.6613.8)
68.4% (68.4625.8)
ES5
2006
17.4%
15.6%
31.5% 29.5%
NA
52.9% (14.268.7)
54.5% (63.7622.9)
57.9% (59.8629.2)
ES6
2006
34.9%
24.8%
36.4% 24.8%
32.8% (34.1618.8)
55.7% (52.5621.8)
44.5% (51.8620.5)
45.2% (52.7619.4)
EBV-associated dead EM3
2001
16.7%
NA
NA
NA
25.2% (19.768.9)
NA
48.3% (56.3623.2)
NA
EM4
2002
18.7%
NA
NA
NA
22.9% (31.6612.8)
NA
65.4% (67.4627.8)
NA
EM5
2005
25.9%
NA
NA
NA
25.2% (19.768.9)
NA
44.7% (63.5629.5)
NA
EM6
2005
12.7%
NA
NA
NA
22.9% (31.6612.8)
NA
57.6% (42.0617.9)
NA
2003
29.4%
24.1%
32.7% 22.8%
61.8% (58.7627.8)
63.5% (60.4629.5)
67.4% (42.1624.8)
47.3% (42.4618.5)
2005
32.1%
NA
30.2% NA
82.1% (74.2635.7)
72.4% (68.5634.7)
85.4% (68.2634.7)
83.4% (57.7632.7)
2006
41.6%
32.5%
37.4% NA
NA
NA
NA
NA
2008
38.5%
28.5%
42.1% NA
56.1% (54.5619.7)
68.7% (62.5634.7)
72.7% (67.5633.2)
78.6% (75.1634.4)
2011
36.9%
NA
30.4% NA
74.3% (64.8631.2)
75.2% (65.4639.2)
86.7% (64.8639.2)
76.2% (74.0632.7)
2005
34.0%
18.4%
44.1% 33.4%
58.4% (48.2623.4)
78.5% (68.4627.9)
84.3% (60.4629.5)
68.8% (60.4629.5)
2007
42.1%
34.2%
38.5% NA
NA
NA
NA
NA
HMB-CAEBV H1
H2
H3
H4
Control* (n = 14)
2012
37.9%
28.1%
35.7% NA
61.5% (54.1628.2)
58.7% (64.2634.2)
52.8% (64.3634.2)
62.7% (70.2632.4)
2005
29.2%
27.4%
25.7% 23.8%
NA
74.8% (65.8627.3)
79.6% (68.0632.9)
72.4% (57.9627.4)
2006
28.7%
NA
27.4% NA
56.1% (54.5619.7)
68.7% (62.5634.7)
72.7% (67.5633.2)
78.6% (75.1634.4)
2011
34.1%
22.5%
40.8% 24.4%
74.3% (64.8631.2)
75.2% (65.4639.2)
86.7% (64.8639.2)
76.2% (74.0632.7)
2006
28.7%
24.5%
32.4% 23.5%
56.4% (42.5615.4)
52.3% (39.4617.9)
75.1% (49.4627.4)
68.7% (43.8614.3)
2010
35.2%
NA
28.2% NA
NA
NA
NA
NA
2012
48.7%
34.9%
34.7% NA
75.7% (54.8627.5)
81.5% (65.3637.8)
68.2% (67.4629.7)
74.8% (72.1638.1)
26.1– 58.9%
20.4– 52.9%
26.1– 20.4– 58.9% 52.9%
54.7–95.2%
42.9–87.4%
Abbreviations: NA, not available. Bold and italicized numbers meant below the normal range. *Healthy normal ranges were obtained from the mean 62 standard deviations. doi:10.1371/journal.pone.0076711.t004
Histiocyte Society [15] and all had hemophagocytosis in bone marrow aspirates. For evidence of EBV infection, EBV RT-PCR detection and viral load detected by copy numbers were determined as previously reported [41]. Serologic antibodies, including anti-viral capsid antigen IgG (EBV-VCA IgG), anti-early antigen IgG (EBEA IgG), anti-viral capsid antigen IgM (EBVVCA IgM), and anti-nuclear antigen (EBNA) were evaluated using immuno-fluorescent ELISA. The clinical features, treatment, prognosis, and immunologic function, including immunoglobulin levels and lymphocyte subsets of T-, B-, NK-, activated lymphocytes, and memory cells, were evaluated and compared in patients with HMB and HLH episodes after Chang Gung Human Investigation Committee approved this study and documented the humanity process. The patients’ parents or guardians provided written and verbal informed consent.
The attenuated perforin expression and predominant-CD3 lymphocyte EBV virus load are distinct in HLH episodes from the HMB episodes. And, the absence of elevated memory CD4+ cell or activated CD2+HALDR+ lymphocytes increase mortality in HLH episodes. Such immunologic alternation rather than genetic defects highlight the possible evolution mechanism of EBVassociated HMB episodes progressing into HLH episodes in the rare cases and warrants further verification through larger-scale studies.
Materials and Methods Patients Patients with an HMB episode had high fever, intense local erythematous responses to mosquito bites, lymphadenopathy, and splenomegaly [21,22]. Those with fulminant HLH episode met the updated diagnostic criteria of the HLH Study Group of the
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Distinction in EBV-Associated HBM and HLH Episodes
permeabilized cells were then incubated in 50 ml staining buffer with 2 ml phycoerythrin (PE)-conjugated anti-perforin and antigranzyme mAb (PharMingen) for 30 min at 4uC. Data from threecolor flow cytometry were calculated and analyzed using the cellquest software (Becton Dickinson).
Cytotoxicity to K562 Leukemia Cell Lines by Single-cell Level Heparinized venous blood samples (10–15 ml) from enrolled patients and healthy controls were delivered to the laboratory within 72 hours. Peripheral blood mononuclear cells (PBMC) as effector cells were isolated from heparinized venous blood by Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ). Effector (E) and K562 target (T) leukemia cells were added in 10 mm610 mm wells to yield E:T ratios of 25:1 and 12.5:1 as indicated if there were enough cells. Control wells, including isolated target or effector cells, were assayed to determine spontaneous cell death. The cells were mixed by gentle tapping, and then centrifuged at 2006g for 1 min and incubated at 37uC in 5% CO2 over night (around 16 hours). Mouse anti-human CD45 monoclonal antibody (ml) directly conjugated with FITC (Pharmingen, San Jose, CA) were added to each tube, mixed gently, and incubated for 20 min on ice. Twenty ml of PI (Sigma, St Louis, MA) at 1 ug/ ml were added to each tube before acquisition. Cytotoxicity to K562 cell lines was measured as the percentage of PI-stained K562 cells by flow cytometry, as previously described [42,43].
Sequence Analysis of SH2D1A/SAP, PFR1, Mun13-4, STX11, STXBP2, XIAP and ITK Genes Total RNA was isolated from PBMC with TRIzol (GIBCOBRL, Gaithersburg, MA) as previously described [44]. Briefly, 2 ug of RNA in a total volume of 20 uL was reverse-transcribed into cDNA using oligo-dT primer and superscript RNaseHreverse transcription (GIBCO-BRL). Two oligonucleotide primers designed from the Gene Bank were selected for each gene to cover the entire coding region of these genes, as previously described [16–20,43,44]. If a specific mutation was identified, the corresponding genomic exons/intron regions were amplified and reconfirmed.
Acknowledgments
Perforin and Granzyme Expression by Flow Cytometry
The authors wish to thank the patients and their families for their kind cooperation, and all of the doctors for their referrals.
Fresh PBMC (56105) were incubated for 30 min at 4uC in 50 ml of staining buffer (PharMingen) supplemented with 10% pooled human serum and 2 ml per CP-conjugated anti-TCRab, fluorescein isothiocyanate (FITC)-conjugated anti-CD16, and/or antiCD56 monoclonal antibody (all from Pharmingen). The cells were then washed, pelleted, and permeabilized in Cytofix/Cytoperm solution (PharMingen) for 20 min at 4uC. The fixed or
Author Contributions Conceived and designed the experiments: W-IL J-LH. Performed the experiments: W-IL J-JL. Analyzed the data: J-JL M-YH S-JL. Contributed reagents/materials/analysis tools: J-JL T-HJ S-HC I-JH C-PY C-JC Y-CH S-PL. Wrote the paper: W-IL.
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